Investigation of the molecular reactivity of bioactive oxiranylmethyloxy anthraquinones

Article abstract of DOI:10.1002/ardp.201900030

The design of a multitarget and multifunctional small molecule containing two functional groups reacting through different mechanisms represents an attractive goal for the medicinal chemist. The preparation of two bifunctional oxiranylmethyloxy anthraquinones, previously investigated as anticancer agents, is described here. These compounds combine a planar, DNA-intercalating and pro-oxidant anthraquinone scaffold and the alkylating epoxide functions which can covalently react with the nucleic acid. Their multilevel molecular reactivity was studied through a combination of analytical techniques: The DNA-binding properties were investigated using a mass spectrometry-based binding assay and by nuclear magnetic resonance, highlighting the formation of a covalent adduct with a nucleobase. Moreover, the contribution of the pro-oxidant redox cycling was evaluated.

Full text of DOI:10.1002/ardp.201900030

Received: 28 January 2019
Revised: 22 March 2019
Accepted: 25 March 2019
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DOI: 10.1002/ardp.201900030
F U L L P A P E R
Investigation of the molecular reactivity of bioactive
oxiranylmethyloxy anthraquinones
Giovanni Ribaudo1
Marco Redaelli1
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Alberto Ongaro2
Giuseppe Zagotto1
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Maira Zorzan3
Maurizio Memo2
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Raffaele Pezzani4
Alessandra Gianoncelli2
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1Department of Pharmaceutical and
Abstract
Pharmacological Sciences, University of
Padova, Padova, Italy
The design of a multitarget and multifunctional small molecule containing two
functional groups reacting through different mechanisms represents an attractive
goal for the medicinal chemist. The preparation of two bifunctional oxiranylmethyloxy
anthraquinones, previously investigated as anticancer agents, is described here.
These compounds combine a planar, DNA‐intercalating and pro‐oxidant anthraqui-
none scaffold and the alkylating epoxide functions which can covalently react with
the nucleic acid. Their multilevel molecular reactivity was studied through a
combination of analytical techniques: The DNA‐binding properties were investigated
using a mass spectrometry‐based binding assay and by nuclear magnetic resonance,
highlighting the formation of a covalent adduct with a nucleobase. Moreover, the
contribution of the pro‐oxidant redox cycling was evaluated.
2Department of Molecular and Translational
Medicine, University of Brescia, Brescia, Italy
3Department of Molecular Medicine (DMM),
University of Padova, Padova, Italy
4O.U. Endocrinology, Department of Medicine
(DIMED), Padova, Italy
Correspondence
Alessandra Gianoncelli, Department of
Molecular and Translational Medicine,
University of Brescia, Viale Europa 11, 25123
Brescia, Italy.
Email: alessandra.gianoncelli@unibs.it
K E Y W O R D S
anthraquinones, anticancer, mass spectrometry, molecular mechanism, redox
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1
INTRODUCTION
set of bis‐(2,3‐epoxypropylamino) anthraquinones, together with
their epoxypropyloxy analogues, and evaluated their activity against
Hydroxylated anthraquinones and their semisynthetic derivatives are
known for their potential as anticancer agents since decades.[1] These
compounds are thought to express their cytotoxic effect through a
combination of several molecular mechanisms, such as topoisome-
rase and protein kinase inhibition and direct interaction with nucleic
acids.[1,2] Moreover, the redox cycling allowed by the presence of the
quinone core results in the promotion of oxidative stress, which may
contribute to the biological activity.[3]
several cancer cell lines, building a preliminary structure–activity
relationship study. Moreover, Pors et al.[6] described the synthesis of
cross‐linkers based on the amino‐anthraquinone scaffold, providing
preliminary activity data on human ovarian cancer A2780 cells. While
in vitro and in vivo cytotoxicity data were previously reported for
these and other compounds from the same class,[5,7] no direct
investigation on the mechanism(s) by which they act at the molecular
level is available in the literature. Two different positional isomers
were synthesized and studied to evaluate the role of the three‐
dimensional arrangement of the molecule in correctly directing the
reacting groups in proximity of nucleotides while interacting with
DNA (Scheme 1). Their multilevel reactivity was tested taking
advantage of a combination of analytical techniques. More in detail,
through a mass spectrometry‐based binding assay, the binding
affinity toward a double‐stranded DNA (dsDNA) sequence was
quantified. The reactivity of the oxiranylmethyloxy moiety of these
potentially cross‐linking agents with a nucleobase, and the subse-
quent formation of the covalent adducts were further investigated by
The idea of combining two or more functional groups with
different mechanisms of action in a single molecule represents a
captivating strategy for drug designers. Concerning the class of
molecules described in this paper, monosubstituted and disubstituted
epoxy anthraquinones have been previously studied as antifungal
and antimicrobial agents.[4] Although, the coexistence of the planar,
DNA‐intercalating anthraquinone scaffold and of the alkylating
epoxide functions which may react covalently with the nucleic acid
made this class of compounds extremely suitable to inspire the
design of anticancer small molecules.[5] Johnson et al.[5] synthesized a
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Arch. Pharm. Chem. Life Sci. 2019;e1900030.
wileyonlinelibrary.com/journal/ardp
© 2019 Deutsche Pharmazeutische Gesellschaft
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O
(a)
OH
O
O
O
O
O
O
i
ii
OH
O
O
O
O
O
O
1,5-dihydroxy anthraquinone
1
2
(b)
O
O
OH
OH
O
O
O
O
O
O
i
ii
O
O
O
1,4-dihydroxy anthraquinone
3
4
(c)
O
H
O
SCHEME 1 Synthetic routes for the
preparation of compounds 2 from 1,
5‐dihydroxy anthraquinone (a) and of 4
from 1,4‐dihydroxy anthraquinone (b). Step
(i): K2CO3, allyl bromide, acetone, reflux.
Step (ii): meta‐chloroperbenzoic acid,
CH2Cl2, room temperature. The reaction
between an oxiranylmethyloxy
N
H2N
O
O
N+
O
O
O
N
DNA
OH
N
DNA
O
O
O
O
anthraquinone and DNA is also
O
2
presented (c)
a kinetic NMR study. Moreover, NMR was also used to gain insights
on the third potentially cytotoxic, redox‐based mechanism of such
molecules, consisting in the pro‐oxidant activity granted by the
anthraquinone core. A compound with limited cytotoxicity, anthrar-
ufin (1,5‐dihydroxy anthraquinone), was selected as a reference
compound in the NMR‐based reactivity investigation.
benzoic acid in CH2Cl2 at room temperature. According to the
literature describing the formation of the epoxide moiety by using
meta‐chloroperbenzoic acid, the products 2 and 4 should be
considered as racemic mixtures.[10–12] Moreover, they cannot be
distinguished by 1H‐NMR under our experimental conditions
(Figures S2 and S4). Thus, the final compounds synthesized
following the procedure described in the Experimental section
were tested as
a racemic mixture. Intermediates and final
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2
RESULTS AND DISCUSSION
Synthesis of the oxiranylmethyloxy
compounds were isolated and analyzed by high‐resolution mass
spectrometry and NMR, and the purity was assessed by high‐
performance liquid chromatography (HPLC). As stated in the
introduction, these compounds and analogues from the same class
were previously tested for their activity as anticancer agents. In
particular, concerning the available bioactivity data, compound 4
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2.1
anthraquinones
The straightforward and efficient preparation of the compounds
was carried out according to the synthetic routes reported in
Scheme 1. The procedures were adapted from the methods
previously reported by Cambie et al.[8] and Brown et al.[9] Briefly,
the first step for the preparation of compounds 2 and 4 was
performed reacting at reflux the opportune dihydroxy anthraqui-
none with allyl bromide in the presence of K2CO3 using acetone as
solvent. The following step was carried out using meta‐chloroper-
was previously tested in
a human tumor cytotoxicity panel
including leukemia, non‐small‐cell lung cancer, colon cancer,
central nervous system cancer, melanoma, ovarian cancer, renal
cancer, prostate cancer, and breast cancer cell lines with promising
results.[5] Further details have also been reported concerning the
bioactivity of these compounds on L1210 leukemia cells.[7]

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2.2
Mass spectrometry binding assay
the cone to 120 V. The variation of this parameter promptly
promoted the appearance of DNA fragments at a lower molecular
weight and charge state. Although, the signal attributed to the DNA‐
compound 2 complex retained similar relative intensity (Figure 1b),
suggesting the presence of an interaction of a stronger nature
between the ligand and the target sequence. Compound 4 showed a
lower tendency to interact with dsDNA under these experimental
conditions (BA = 21.4 at 80 V, Figure S12).
Compounds 2 and 4 were tested for their binding capability towards
dsDNA in gas phase using high‐resolution mass spectrometry.[13,14]
The dsDNA resulting from the annealing of single strands 5′‐ACT
ATT CCC GGG TAA TGA‐3′ and 5′‐TCA TTA CCC GGG TAA AGT‐3′,
which represents a model of relevant biological interest for many
aspects, was used as a target for this test. In fact, DNA sequences
containing core palindromic GC‐rich hexanucleotides were detected
within several oncogenes in the human genome. Moreover, they have
been identified in peculiar regions of HIV‐1 retrovirus genome.[15–17]
As depicted in Figure 1, it was possible to observe that compound
2 interacts with dsDNA forming 1:1 and 2:1 complex. The stability of
the dsDNA–ligand complex was consequently investigated tuning the
cone voltage parameter. “Cone voltage driven dissociation” is one of
the approaches to measure interaction strengths, and, in particular,
the increase of voltage (usually among the 100–220 V range) is
commonly used to promote the complex dissociation in gas phase
high‐resolution mass spectrometry binding experiments.[18,19] First,
the sample was acquired setting the sample cone at 80 V in negative
ionization mode, and the z = 6 charge state was considered, in which
the complex peak was detectable (Figures S10 and S11 show the
peaks corresponding to the 1:1 complex, highlighting the presence of
a sodium adduct at m/z = 1893). Compound 2 showed a good
tendency to bind the duplex DNA sequence, with a calculated binding
affinity (BA) of 41.8.[20,21] The sample was then reacquired setting
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2.3
NMR‐assisted study of the ligand–guanine
reaction mechanism
Guanine has been previously shown to be the main site of reaction
for epoxides with DNA, and preliminary studies on the reactivity of
simple epoxides demonstrated that about 80% of reaction
products on DNA are 7‐alkylguanines.[22,23] Hansen et al. studied
the behavior of hedamycin, a more complex molecule belonging to
a class of antibiotics that achieve their activity by intercalating
DNA, and subsequently alkylating the macromolecule in an
epoxide‐mediated manner. The authors investigated, using NMR
spectroscopy, the structure of the drug‐DNA complex and, in
particular, highlighted the change in the chemical shift of H8 signal
of guanine once the reaction occurred.[24] Hedamycin was also
studied by Pavlopoulos et al.[25], who confirmed the crucial role of
guanine as nucleophilic site.
FIGURE 1 MS spectra showing the interaction between the dsDNA sequence and compound 2 (z = 6, negative ionization mode). Setting the
sample cone to 80 V, the spectrum clearly showed the presence of 1:1 and 2:1 DNA‐ligand complexes with ammonia adducts (a). At higher
voltage values, other DNA fragments appear on the spectrum but the 2:1 complex is still clearly present (120 V) (b). dsDNA: double‐stranded
DNA; MS: mass spectrometry

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At neutral pH, the reactivity of epoxides is reported to follow a
SN2 mechanism.[24,26] Acid‐catalyzed epoxide ring opening is a
nucleophilic substitution reaction that substantially occurs with a
SN1 character. The protonation at the oxygen atom promotes the
formation of the carbocation of the most substituted carbon atom,
that can become the site of attack of the nucleophile.[27] Acidic
conditions have been previously adopted by other authors to
promote model reactions between epoxides and guanine.[25] Johnson
et al. observed an increase in the rate of hydrolysis of aflatoxin B1
exo‐8,9‐epoxide by calf thymus DNA following acid catalysis (pH <
5.4), but the same reaction was not base‐catalyzed.[28,29]
catalysis, as discussed above, and to promote the dissolution of
guanine, which is poorly soluble, in the reaction environment.
Moreover, TFA does not interfere with NMR analysis. Under the
NMR study conditions, two guanine molecules conducted
a
nucleophilic attack toward the most substituted carbon atoms of
the acid‐activated epoxide rings. Compounds 2 and 4 reacted
similarly toward guanine (Figures
2 and 3), leading to the
formation of the double adducts, which give the characteristic
signals at 3.6, 3.9, and 4.1 ppm in the proton spectrum. The
formation of such molecules was also accompanied by the
production, and the subsequent consumption, of monosubstituted
intermediates which gave a signal at 4.6 ppm in the proton
spectrum that disappeared at the end of the reaction. After 60–
70 min since the beginning, the signals corresponding to the
starting epoxide were barely detectable in the spectrum.
In the current study, the reaction between compound 2 (and 4)
and the nucleobase was monitored in real time using NMR
spectrometry. The experiments were carried out in NMR sample
tubes in dimethyl sulfoxide (DMSO)‐d6 with 5% trifluoroacetic
acid (TFA), according to the procedure reported in the experi-
mental section. TFA was introduced to take advantage of the acid
To avoid any bias in the interpretation of the NMR results
concerning the reactivity of the epoxides, a control reaction was
FIGURE 2 Superimposition of the NMR spectra recorded at different time points to monitor the reaction between oxiranylmethyloxy
anthraquinones and guanine. The reaction of compound 2 (a) and compound 4 (b) proceed similarly to the formation of the N‐alkyl derivative
with almost complete disappearance of the starting molecule. NMR: nuclear magnetic resonance

RIBAUDO ET AL.
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FIGURE 3 Kinetic plots for the
reactions of guanine with compound 2
(a) and with compound 4 (b). As discussed
above, compounds 2 and 4 react with the
nucleobase forming a monosubstituted
intermediate and the product. In both the
reactions, the relative abundance of
monosubstituted intermediates never
exceeded 10–12%. The rates of
disappearance of the starting compounds
are similar, there are instead differences in
the rates of formation of the products: The
relative abundance of double‐reacted
compound 2 depends linearly on the time
whereas the behavior of compound 4 is
close to a sigmoid
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performed under the same experimental conditions by dissolving
1 mg/ml oxiranylmethyloxy anthraquinone in 600 µl of DMSO‐d6 and
5% TFA, without addition the nucleobase as a reacting nucleophile.
As demonstrated by the NMR monitoring, this reaction slowly
proceeds to the formation of the diol (Figure S16). Moreover, 1,
5‐dihydroxy anthraquinone was also reacted under similar conditions
with guanine as a negative control. Anthrarufin (1,5‐dihydroxy
anthraquinone) was selected as a reference compound in this test
because it represents the starting material for the preparation of
compound 2, and was reported to show limited cytotoxicity.[30,31]
This compound was also previously investigated as an antioxidant
and an anti‐inflammatory agent.[32] As expected, since the compound
lacks the reactive moieties, no interaction was observed with guanine
in solution (Figure S17). Finally, as a further confirmation, a mass
spectrometric analysis of the oxiranylmethyloxy anthraquinone‐
guanine reaction was carried out in positive ionization mode, which
demonstrated the formation of a covalent adduct (Figures S18
and S19).
2.4
NMR study of the reaction with Na2S2O4
Redox kinetics may play a role in the observed biological effects of
pro‐oxidant, cytotoxic species. An NMR‐based experiment was
performed to highlight the role of the position of the substituents
in the anthraquinone scaffold within the redox cycling. The reactivity
in solution of compounds 2 and 4 toward Na2S2O4, a reducing agent,
was measured by NMR.[33,34] Compound 2 proved to be a better pro‐
oxidant by reacting promptly with 1 equivalent of Na2S2O4, being
partially converted to the reduced species (30%, Figures S20 and
S21). Although, the compound showed a high tendency to quickly
reconvert to its oxidized form. No detectable reactivity was observed
for compound 4 under the same experimental conditions, while both
compounds showed complete conversion to the reduced form after
24 hr from the addition of three equivalents of Na2S2O4
(Figures S22–S24). Again, anthrarufin was introduced in the experi-
mental routine as a reference compound. This hydroxy anthraqui-
none showed a very good tendency to react with Na2S2O4, being

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FIGURE 4 Computed 3D interaction models for the interaction of compounds 2 (a) and 4 (b) with a dsDNA sequence. dsDNA: double‐
stranded DNA; 3D: three‐dimensional
promptly converted to the reduced species in the presence of one
equivalent of the reducing agent (Figure S25). Although, this
reactivity is not paralleled by the capability of forming a covalent
interaction with the nucleobase. Thus, the compound may not rely on
the synergistic mechanisms exploited by the oxiranylmethyloxy
anthraquinones.
characterized using a combination of analytical techniques, demon-
strating that both compounds 2 and 4 bind to isolated guanine in
solution. Compound
2 gave a stable interaction with dsDNA,
suggesting a relevant role of the three‐dimensional arrangement of
the molecule in correctly directing the reacting groups, after the
intercalation, towards the nucleophilic sites of dsDNA. This was also
confirmed by a preliminary computational study investigating the
binding motif towards dsDNA. The results of the NMR‐based redox
study highlighted that compound 2 exceeds compound 4 also in
terms of pro‐oxidant activity.
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2.5
Computational studies
The interaction pattern between compounds 2 and 4 and DNA was
investigated in silico. As can be observed in the 3D models
represented in Figure 4, when bound to dsDNA, the reactive groups
on the side chains of compound 2 are in closer proximity to the
nucleophilic guanine. According to the computed model, the 1,5‐
disubstituted anthraquinone shows a conformation which better
allows the interaction of the alkylating functions with the
nucleic acid.
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4
EXPERIMENTAL
Chemistry
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4.1
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4.1.1
General
Commercially available chemicals were purchased from Sigma‐
Aldrich (St. Louis, MO) and used as received, unless otherwise
stated. 1H‐ and 13C‐NMR spectra were recorded on a Bruker
Avance III (Bruker, Billerica, MA) 400 MHz spectrometer. The
solvent for each spectrum is given in parentheses. Chemical shifts
are reported in ppm and are relative to tetramethylsilane (TMS)
internally referenced to the residual solvent peak. Datasets were
edited with Bruker TopSpin software (Bruker). The multiplicity of
signals is reported as singlet (s), doublet (d), triplet (t), quartet (q),
multiplet (m), broad (br), or a combination of any of these. High‐
resolution mass spectra were recorded on an Applied Biosystems
(Foster City, CA) Mariner electrospray ionization time‐of‐flight
(ESI–TOF) spectrometer. The purity profile (above 96% unless
otherwise stated) was assayed by HPLC using a Varian Pro‐Star
system equipped with a Bio‐Rad (Hercules, CA) 1706 ultraviolet–
visible detector (254 nm) and an Agilent C‐18 column (5 μm,
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3
CONCLUSIONS
The here described oxiranylmethyloxy anthraquinones conjugate
structural features that allow a synergistic effect between multiple
molecular mechanisms of primary relevance for their biological role,
intercalation, alkylation, and redox activity. The results demonstrate
the relevance of these synergistic mechanisms exploited by the
oxiranylmethyloxy anthraquinones. The compounds have been tested
using high‐resolution mass spectrometry for their binding capability
toward a biologically relevant dsDNA sequence, and compound 2
proved to interact more effectively with the nucleic acid. The
strength of the interaction and the binding mechanism were also
investigated. NMR was used to monitor the kinetic of a simplified
model reaction and the formation of the covalent adduct was fully

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4.6 × 250 mm). An appropriate ratio of water (A) and acetonitrile
(B) was used as mobile phase with an overall flow rate of 1 ml/min;
the general method for the analyses is reported here: 0 min (95%
A–5% B), 5 min (95% A–5% B), 15 min (5% A–95% B), 20 min (5%
A–95% B), and 22 min (95% A–5% B).
4.1.5
Synthesis of 1,4‐di(oxiranylmethyloxy)‐
anthraquinone (4)
The compound was synthesized following the procedure described for
compound 2. Yield: 68%. δH (400 MHz, DMSO) 2.87 (d, 2H, CH), 2.92 (d,
2H, CH), 3.42 (d, 2H, CH), 4.07 (dd, 2H, CH), 4.47 (dd, 2H, CH), 7.56 (s,
2H, ArH), 7.84 (m, 2H, ArH), 8.05 (m, 2H, ArH). δC (100 MHz, DMSO)
44.1, 50.3, 70.8, 122.9, 123.3, 126.4, 134.1, 134.2, 158.3, 182.4. HMRS
(ESI) found 353.1018 (C20H17O6, [M+H]+), calc. 353.1020.
The InChI codes of compounds 2 and 4 are given in the
Supporting Information.
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4.1.2
Synthesis of 1,5‐bis(prop‐2′‐enyloxy)‐
anthraquinone (1)
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4.2
Mass spectrometry binding assay
The compound was synthesized according to a procedure previously
reported by Cambie et al.[8] with slight modifications. A round‐
bottomed flask was charged with 1,5‐dihydroxy anthraquinone
(2.08 mmol) and 75 ml of acetone. K2CO3 (25.40 mmol) and allyl
bromide (23.20 mmol) were then added and the mixture was heated
at reflux for 2 days. The hot mixture was filtered, and the filtrate was
concentrated under reduced pressure. The resulting solid was
dissolved in 100 ml of CH2Cl2 and washed with water. The organic
layer was then evaporated providing the desired product as a dark
yellow solid. Yield: 87%. δH (400 MHz, CDCl3) 4.87 (d, 4H, CH2), 5.41
(dd, 2H, CH), 5.68 (dd, 2H, CH), 6.16 (m, 2H, CH), 7.26 (d, 2H, ArH),
7.69 (t, 2H, ArH), 7.94 (d, 2H, ArH). HMRS (ESI) found 321.1125
(C20H17O4, [M+H]+), calc. 321.1121.
DNA substrates (single strand 1: 5′‐ACT ATT CCC GGG TAA TGA‐3′,
exact mass 5495.95 Da; single strand 2: 5′‐TCA TTA CCC GGG TAA
AGT‐3′, exact mass 5495.95 Da) were obtained from Sigma‐Aldrich. The
preparation of the samples for the binding experiment was carried out
according to a previously reported procedure.[21] The oligonucleotides
were diluted to final concentration of 5 μM and incubated with the
tested compound at a DNA/compound ratio of 1:5 overnight at room
temperature. Samples were analyzed by direct infusion electrospray
ionization (ESI, negative ionization mode) on a Waters (Milford, MA)
Xevo G2‐XS QTof mass spectrometer using 100 mM ammonium acetate
as carrying buffer. Five microliters of samples were typically injected for
each analysis. The electrospray capillary was set at 1.8 kV, the source
and desolvation temperatures were set to 45°C and 65°C, respectively.
All these parameters ensured minimal fragmentation of the DNA
complexes. The sampling cone was set to 80 or 120 V, as specified for
the different experiments. The instrument was calibrated using a 2 mg/
ml solution of sodium iodide in 50% isopropanol. Additionally, the use of
the LockSpray™ technology during the analysis provided a typical <2
ppm mass accuracy. Exact mass for the dsDNA: 10991.90 Da (please
refer to Supporting Information for high‐resolution mass spectra of the
structured dsDNA; Figures S7 and S8). For the test reaction between
the oxiranylmethyloxy anthraquinone and guanine, mass spectra were
recorded on an Applied Biosystems Mariner ESI–TOF spectrometer in
positive ionization mode.
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4.1.3
Synthesis of 1,5‐di(oxiranylmethyloxy)‐
anthraquinone (2)
A round‐bottomed flask was charged with compound 1 (0.16 mmol)
and 10 ml of CH2Cl2. The mixture was stirred at room temperature
under a nitrogen atmosphere and a solution of meta‐chloroperben-
zoic acid (0.62 mmol) in 5 ml of CH2Cl2 was added drop by drop to
the reaction. After 72 hr of stirring, TLC showed complete consump-
tion of the starting material (hexane/ethyl acetate 2:1). The solution
was washed with 20% sodium bisulfite and water. The organic layer
was evaporated providing the desired product as a dark yellow solid.
Yield: 65%. δH (400 MHz, DMSO) 2.89 (d, 2H, CH), 2.98 (d, 2H, CH),
3.42 (d, 2H, CH), 4.13 (dd, 2H, CH), 4.51 (dd, 2H, CH), 7.51 (d, 2H,
ArH), 7.75 (d, 2H, ArH), 7.79 (t, 2H, ArH). δC (100 MHz, DMSO) 44.2,
50.1, 68.8, 119.4, 119.7, 120.8, 135.8, 137.2, 158.7, 181.8. HMRS
(ESI) found 353.1026 (C20H17O6, [M+H]+), calc. 353.1020.
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4.3
NMR‐assisted study of the ligand–guanine
reaction mechanism
The opportune oxiranylmethyloxy anthraquinone was dissolved in
600 µl of DMSO‐d6 (1 mg/ml) in an NMR sample tube. After the
acquisition of the reference 1H spectrum, guanine (four equivalents
from a 20 mg/ml solution in DMSO‐d6 with 5% TFA) was added. The
reaction was carried out inside the Bruker Avance III 400 MHz
spectrometer, acquiring 1H spectra at different time points to follow
the formation of the products. Datasets were edited using Bruker
TopSpin software and manual integration.
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4.1.4
Synthesis of 1,4‐bis(prop‐2′‐enyloxy)‐9,10‐
anthraquinone (3)
The compound was synthesized according to Brown et al.[9] with
slight modifications and following the procedure described for
compound 1. The product was isolated as a yellow solid. Yield:
89%. 4.73 (d, 4H, CH2), 6.39 (dd, 2H, CH), 5.81 (dd, 2H, CH), 6.16
(m, 2H, CH), 7.29 (s, 2H, ArH), 7.72 (m, 2H, ArH), 8.21 (m, 2H,
ArH). HMRS (ESI) found 321.1127 (C20H17O4, [M+H]+), calc.
321.1121.
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4.4
NMR study of the reaction with Na2S2O4
The opportune oxiranylmethyloxy anthraquinone was dissolved in
600 µl of DMSO‐d6 (1 mg/ml) in an NMR sample tube. After the

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acquisition of the reference 1H spectrum, Na2S2O4 (1–3 equivalents
from a 10 mg/ml solution in D2O) was added, according to a
previously reported procedure.[33,34] Data acquisition and processing
were performed as described above.
[12] H. Toda, R. Imae, N. Itoh, Adv. Synth. Catal. 2014, 356, 3443.
[13] R. Ferreira, A. Marchand, V. Gabelica, Methods. 2012, 57, 56.
[14] F. Doria, M. Nadai, M. Folini, M. Scalabrin, L. Germani, G. Sattin, M.
Mella, M. Palumbo, N. Zaffaroni, D. Fabris, M. Freccero, S. N. Richter,
Chem. A Eur. J. 2013, 19, 78.
[15] S. Wain‐Hobson, P. Sonigo, O. Danos, S. Cole, M. Alizon, Cell 1985,
40, 9.
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4.5
Computational studies
[16] A. Gianoncelli, S. Basili, M. Scalabrin, A. Sosic, S. Moro, G. Zagotto, M.
Palumbo, N. Gresh, B. Gatto, ChemMedChem 2010, 5, 1080.
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Pasquale, G. Capranico, G. Ribaudo, M. Palumbo, Arch. Pharm. Chem.
Life Sci. 2014, 347, 728.
The in silico study consisted in the receptor preparation and the
docking simulation, which were conducted with the LeadIT software
(version 2.3.2; BioSolveIT GmbH, Sankt Augustin, Germany, 2017,
www.biosolveit.de/LeadIT). The used macromolecule was a dsDNA
sequence retrieved from the Protein Data Bank, PDB ID: 108D.
[18] A. Kapur, J. L. Beck, S. E. Brown, N. E. Dixon, M. M. Sheil, Protein Sci.
2002, 11, 147.
[19] J. M. Daniel, G. McCombie, S. Wendt, R. Zenobi, J. Am. Soci. Mass
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[20] W. Tan, J. Zhou, G. Yuan, Rapid Commun. Mass Spectrom. 2014,
28, 143.
ACKNOWLEDGMENT
[21] G. Ribaudo, M. Scalabrin, V. Pavan, D. Fabris, G. Zagotto, ARKIVOC
2016, 2016, 145.
This study work was supported by University of Padova and
University of Brescia.
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ORCID
Giuseppe Zagotto
http://orcid.org/0000-0001-7053-4196
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